Synthetic promoters

ABSTRACT

Synthetic elements for enhancing expression of genes in plant cells are disclosed. These include a promoter with a &#34;TATA to start&#34; sequence containing 64% or greater GC content and an synthetic upstream element incorporating several OCS binding motifs and novel flanking sequences. Upstream activating regions (UARs) are also disclosed that can further increase the constitutive transcriptional activity when they are operably linked to said promoter and/or the synthetic upstream element. In particular, the nucleotide sequence of the UAR of the maize Ubi-1 gene is provided and its use in expression cassettes and vectors containing these promoter elements. Cells and plants transformed with these vectors are further provided. These include a transgenic sunflower expressing an exogenous oxalate oxidase gene at a high level under the transcriptional control of a recombinant promoter having at least one upstream activating region of the 35S CaMV promoter.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/661,601, filed on Jun. 11, 1996 now abandoned, hereinincorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of plant molecular biologyand in particular to enhanced expression of desired structural genes inboth monocotyledonous and dicotyledonous plants.

BACKGROUND OF THE INVENTION

Gene expression encompasses a number of steps originating from the DNAtemplate ultimately to the final protein or protein product. Control andregulation of gene expression can occur through numerous mechanisms. Theinitiation of transcription of a gene is generally thought of as thepredominant control of gene expression. The transcriptional controls (orpromoters) are generally relegated to relatively short sequencesimbedded in the 5'-flanking or upstream region of the transcribed gene.There are DNA sequences which affect gene expression in response toenvironmental stimuli, nutrient availability, or adverse conditionsincluding heat shock, anaerobiosis or the presence of heavy metals.There are also DNA sequences which control gene expression duringdevelopment or in a tissue, or organ specific fashion.

Promoters contain the signals for RNA polymerase to begin transcriptionso that protein synthesis can proceed. DNA binding, nuclear proteinsinteract specifically with these cognate promoter DNA sequences topromote the formation of the transcriptional complex and eventuallyinitiate the gene expression process.

One of the most common sequence motifs present in the promoters of genestranscribed by eukaryotic RNA polymerase II (polII) system is the "TATA"element which resides upstream of the start of transcription. Eukaryoticpromoters are complex and are comprised of components which include aTATA box consensus sequence at about 35 base pairs 5' relative to thetranscription start site or cap site which is defined as +1. The TATAmotif is the site where the TATA-binding-protein (TBP) as part of acomplex of several polypeptides (TFIID complex) binds and productivelyinteracts (directly or indirectly) with factors bound to other sequenceelements of the promoter. This TFIID complex in turn recruits the RNApolymerase II complex to be positioned for the start of transcriptiongenerally 25 to 30 base pairs downstream of the TATA element andpromotes elongation thus producing RNA molecules. The sequences aroundthe start of transcription (designated INR) of some polI genes seem toprovide an alternate binding site for factors that also recruit membersof the TFIID complex and thus "activate" transcription. These INRsequences are particularly relevant in promoters that lack functionalTATA elements providing the core promoter binding sites for eventualtranscription. It has been proposed that promoters containing both afunctional TATA and INR motif are the most efficient in transcriptionalactivity. (Zenzie-Gregory et al, 1992. J. Biol. Chem. 267:2823-2830).

In most instances sequence elements other than the TATA motif arerequired for accurate transcription. Such elements are often locatedupstream of the TATA motif and a subset may have homology to theconsensus sequence CCAAT.

Other DNA sequences have been found to elevate the overall level ofexpression of the nearby genes. One of the more common elements thathave been described reside far upstream from the initiation site andseem to exhibit position and orientation independent characteristics.These far upstream elements have been designated enhancers.

One of the less common elements by virtue of their specificities aresequences that interact with specific DNA binding factors. Thesesequence motifs are collectively known as upstream elements which areusually position and orientation dependent.

Many upstream elements have been identified in a number of plantpromoters based initially on function and secondarily on sequencehomologies. These promoter upstream elements range widely in type ofcontrol: from environmental responses like temperature, moisture,wounding, etc., developmental cues, (germination, seed maturation,flowering, etc.) to spatial information (tissue specificity). Theseelements also seem to exhibit modularity in that they may be exchangedwith other elements while maintaining their characteristic control overgene expression.

Promoters are usually positioned 5' or upstream relative to the start ofthe coding region of the corresponding gene, and the entire regioncontaining all the ancillary elements affecting regulation or absolutelevels of transcription may be comprised of less than 100 base pairs oras much as 1 kilobase pair.

A number of promoters which are active in plant cells have beendescribed in the literature. These include nopaline synthase (NOS) andoctopine synthase (OCS) promoters (which are carried on tumor inducingplasmids of Agrobacterium tumefaciens). The cauliflower mosaic virus(CaMV) 19S and 35S promoters, the light-inducible promoter from thesmall subunit of ribulose bisphosphate carboxylase (ssRUBICSO, a veryabundant plant polypeptide), and the sucrose synthase promoter are alsoincluded. All of these promoters have been used to create various typesof DNA constructs which have been expressed in plants. (See for examplePCT publication WO84/02913 Rogers, et al).

Two promoters that have been widely used in plant cell transformationsare those of the genes encoding alcohol dehydrogenase, AdhI and AdhII.Both genes are induced after the onset of anaerobiosis. Maize AdhI hasbeen cloned and sequenced as has been AdhII. Formation of an AdhIchimeric gene, Adh-Cat comprising the AdhI promoter links to thechloramphenicol acetyltransferase (CAT) coding sequences and nopalinesynthase (NOS) 3' signal caused CAT expression at approximately 4-foldhigher levels at low oxygen concentrations than under controlconditions. Sequence elements necessary for anaerobic induction of theADH-CAT chimeric have also been identified. The existence of anaerobicregulatory element (ARE) between positions -140 and -99 of the maizeAdhI promoter composed of at least two sequence elements positions -133to -124 and positions -113 to 99 both of which have found to benecessary and are sufficient for low oxygen expression of ADH-CAT geneactivity. The Adh promoter however responds to anaerobiosis and is not aconstitutive promoter drastically limiting its effectiveness.

Another commonly used promoter is the 35S promoter of Cauliflower MosaicVirus. The (CaMV) 35S promoter is a dicot virus promoter however itdirects expression of genes introduced into protoplasts of both dicotsand monocots. The 35S promoter is a very strong promoter and thisaccounts for its widespread use for high level expression of traits intransgenic plants. The CaMV35S promoter however has also demonstratedrelatively low activity in several agriculturally significantgraminaceous plants such as wheat. While these promoters all give highexpression in dicots, few give high levels of expression in monocots. Aneed exists for a synthetic promoters and other elements that induceexpression in transformed monocot protoplast cells.

SUMMARY OF THE INVENTION

Methods and compositions for the expression of heterologous sequences inhost cells are provided. The compositions find particular use incontrolling the expression of sequences in plants. The compositions ofthe invention comprise promoter sequences. In particular, a novelsynthetic core promoter molecule and regulatory elements useful incontrolling expression in target cells are provided. The core promotercomprises a TATA box and a start of transcription. Further, the "TATA tostart" region is 64% or greater GC rich. The regulatory elements includea novel upstream element and upstream activating regions. The upstreamactivating region is different from the synthetic upstream element. Theelements can be used together or with other promoter elements to controlexpression of sequences of interest.

It is a primary object of the invention to provide synthetic regulatoryelements that enhance expression of introduced genes in plant cells andplant tissues.

It is an object of the invention to provide a recombinant promotermolecule that provides for reliably high levels of expression ofintroduced genes in target cells. It is yet another object of theinvention to provide heterologous upstream enhancer elements that canenhance the activity of any promoter.

It is yet another object of the invention to provide plants, plant cellsand plant tissues containing either or both of the recombinant promoteror upstream element of the invention.

It is yet another object of the invention to provide vehicles fortransformation of plant cells including viral or plasmid vectors andexpression cassettes incorporating the synthetic promoter and upstreamelements of the invention.

It is yet another object of the invention to provide bacterial cellscomprising such vectors for maintenance, and plant transformation.

Other objects of the invention will become apparent from the descriptionof the invention which follows.

DESCRIPTION OF THE FIGURES

FIG. 1 is a depiction of a typical nucleotide base arrangement of a corepromoter containing the consensus sequences of TATA and INR motifspresent in plant promoters. A designates +1 of the transcribed region.

FIG. 2 is a depiction of the complete Syn II Core Promoter Sequence withan example of a plant promoter and both are aligned at the major startof transcription (bold letter). The TATA motif is underlined. The CaMV35S promoter is shown with percent GC content sequences shown inparentheses.

FIG. 3 is the DNA sequence of the Rsyn 7 upstream element. The TGACGmotifs are indicated in bold.

FIG. 4 is a plasmid map of one embodiment of the invention comprisingthe Syn II Core promoter and Rsyn7 elements driving a GUS containingconstruct.

FIG. 5 depicts several schematics of synthetic promoters according tothe present invention tested in transient and stable transformants.

FIG. 6 is a depiction of transient assay data using the plasmidsincorporating the promoter sequences of the invention.

FIG. 7(A). Rsyn7::GUS (PHP6086) activity to T0 maize plants.

FIG. 7(B) is a schematic of VT stage corn plants with sites of tissuesamples indicated.

FIG. 8 depicts GUS activity in root segments of a segregating populationof maize T1 transgenic seedlings containing the Rsyn7::GUS (PHP6086) orthe UBI:GUS (PHP3953) construct.

FIG. 9 depicts GUS expression of three synthetic promoters in T0transgenic maize plants including the promoter sequences of theinvention as comparison.

FIG. 10 shows the comparison of the activities of the Rsyn7 promoter,the CaMV 35S promoter, and the 35SU-Rsyn7 promoter in transientexpression in sunflower cotyledons.

FIG. 11 shows the effect of the Ubi-1 upstream activating region on thestrength of the Rsyn 7 promoter in transient expression in sunflowercotyledons.

FIG. 12 shows that in the stably transformed sunflower callus, GUSexpression behind the control of the 35SU-Rsyn7 is 20% higher than whenbehind the control of the 35S CaMV promoter.

FIG. 13 shows the effect of the Ubi-1 upstream activating region on theactivity of the Rsyn7 promoter in transgenic sunflower callus assay.

DETAILED DESCRIPTION OF THE INVENTION

In the description that follows a number of terms are used extensively.The following definitions are provided in order to remove ambiguities inthe intent or scope of their usage in the specification and claims, andto facilitate understanding of the invention.

A structural gene is a DNA sequence that is transcribed into messengerRNA (mRNA) which is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

A promoter is a DNA sequence that directs the transcription of astructural gene. Typically a promoter is located in the 5' region of agene, proximal to the transcriptional start site of a structural gene.The promoter of the invention comprises at least a core promoter asdefined below. Additionally, the promoter may also include at least oneupstream elements. Such elements include UARs and optionally, other DNAsequences that affect transcription of a structural gene such as asynthetic upstream element.

A core promoter or minimal promoter contains the essential nucleotidesequences for expression of the operably linked coding sequence,including the TATA box and start of transcription. By this definition, acore promoter may or may not have detectable activity in the absence ofspecific sequences that may enhance the activity or confer tissuespecific activity. For example, the maize SGB6 gene core promoterconsists of about 37 nucleotides 5' of the transcriptional start site ofthe SGB6 gene, while the Cauliflower Mosaic Virus (CaMV) 35S corepromoter consists of about 33 nucleotides 5' of the transcriptionalstart site of the 35S genome.

ADH refers generally to a plant expressible alcohol dehydrogenase geneand specifically to the alcohol dehydrogenase gene from maize.

ADH 1 UAR refers to the DNA fragment spanning the region betweennucleotide positions about -1094 to about -106 of the alcoholdehydrogenase gene 1 from maize, or homologous fragment that isfunctionally equivalent. The sequence is numbered with the start oftranscription site designated as +1 according to the correctionpublished by Ellis et al. (1987) supra.

"TATA to start" shall mean the sequence between the primary TATA motifand the start of transcription.

A synthetic DNA is an artificially created DNA sequence that is notproduced naturally, and must be introduced to an organism or to anancestor of that organism to control or to be expressed.

OCS element refers to the TGACG motif identified from the octopinesynthase gene, histone genes, enzyme genes for agropine biosynthesis,the mannopine synthase gene, the CaMV 35S gene, histone H3 gene andnopaline synthase gene. As used herein the term includes any sequencecapable of binding the ASF-1 factor as identified in U.S. Pat. No.4,990,607 by Katagiri, the disclosure of which is incorporated byreference.

UAR is typically a position or orientation dependent element thatprimarily directs tissue, cell type, or regulated expression.

An enhancer is a DNA regulatory element that can increase efficiency oftranscription regardless of the distance or orientation of the enhancerrelative to the start site of transcription.

The term expression refers to biosynthesis of a gene product. In thecase of a structural gene, expression involves transcription of thestructural gene into mRNA and then translation of the mRNA into one ormore polypeptides.

A cloning vector is a DNA molecule such as a plasmid, cosmid orbacterial phage that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss ofessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance, hygromycin resistance orampicillin resistance.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically gene expression is placed under thecontrol of certain regulatory elements including promoters, tissuespecific regulatory elements, and enhancers. Such a gene is said to be"operably linked to" the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or an expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned genes in the chromosome orgenome of the host cell.

A transgenic plant is a plant having one or more plant cells thatcontain an expression vector.

It will be understood that there may be minor sequence variations withinsequence or fragments used or disclosed in this application. By "minorvariations" is intended that the sequences have at least 80%, preferably90% sequence identity. These variations may be determined by standardtechniques to enable those of ordinary skill in the art to manipulateand bring into utility the functional units of the promoter elementsnecessary to direct initiation of transcription in the structural genefollowed by a plant expressible transcription termination (and perhapspolyadenylation) signal.

Plant tissue includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

One embodiment of the invention, the core promoter, is shown in SEQ IDNO:1. The core promoter is capable of driving expression of a codingsequence in a target cell, particularly plant cells. The core promoterfinds use in driving expression of sequences which are only needed atminimal levels in the target cells. Also disclosed is a novel upstreamelement, SEQ ID NO:2 that helps to potentiate transcription. Thesynthetic core promoter can be used with combinations of enhancer,upstream elements, and/or activating sequences from the 5'-flankingregions of plant expressible structural genes. Similarly the upstreamelement can be used in combination with various plant core promotersequences. In one embodiment the core promoter and upstream element areused together to obtain ten-fold higher expression of an introducedmarker gene in monocot transgenic plants than is obtained with the maizeubiquitin 1 promoter.

The core promoter comprises a TATA motif and a GC rich "TATA to start oftranscription" region (64% or greater GC content that is generallycharacteristic of animal promoters. The sequence is placed 5' of astructural gene and will promote constitutive expression which isnon-tissue specific in transgenic plant cells.

The invention also comprises an expression cassette comprising (theupstream element) the synthetic core promoter, a structural gene, theexpression of which is desired in plant cells, and a polyadenylation orstop signal. The expression cassette can be encompassed in plasmid orviral vectors for transformation of plant protoplast cells.

The invention also encompasses transformed bacterial cells formaintenance and replication of the vector, as well as transformedmonocot or dicot cells and ultimately transgenic plants.

In another embodiment, the invention encompasses an upstream elementthat can be used in combination with the synthetic promoter or withother known promoters in the art. The upstream element comprises atleast 3 OCS binding motifs (TGACG) with a novel intervening sequence.One embodiment is disclosed in SEQ ID NO:2 and is placed 5' to a corepromoter sequence to enhance the transcription levels of the resultinggene product. Thus the invention comprises an expression cassettecomprising the synthetic upstream element of the invention, 5' to aplant inducible promoter which is 5' to a structural gene. Thisexpression cassette can be embodied in vectors and plasmids as earlierdescribed.

In a preferred embodiment the synthetic upstream element is used incombination with the synthetic core promoter sequence to achievenon-tissue specific constitutive expression of the gene product which isa ten-fold enhancement of the maize Ubi-1 promoter.

The present invention also encompasses a promoter construct comprisingthe synthetic core promoter described above and an upstream activatingregion. The upstream activating region is different from the syntheticupstream element. Preferably the upstream activating region is anupstream activating region (UAR) having substantial sequence similarityto the UAR of CaMV 35S or maize Ubi-1. Promoter constructs of theinvention may comprise the synthetic core promoter in combination withat least one UAR and optionally at least one synthetic upstream element.

The promoter construct can be contained for convenience in an expressioncassette. This expression cassette can be embodied in transformationvectors.

The sequence of the upstream activating region (UAR) of the maize Ubi-1gene is also provided. This UAR can be used in combination with any corepromoter to enhance the activity of the promoter.

The promoter of the invention as seen in SEQ ID NO: 1 and/or SEQ IDNO:10 (modified core promoter), can be used to obtain high levels ofexpression of structural genes. Similarly the upstream element of theinvention (SEQ ID NO:2) can be used in combination with other promotersor the promoter of the invention to potentiate levels of transcriptionin genetically modified plants. Production of a genetically modifiedplant tissue expressing a structural gene under the control of theregulatory elements of the invention combines teachings of the presentdisclosure with a variety of techniques and expedients known in the art.In most instances alternate expedients exist for each stage of theoverall process. The choice of expedients depends on the variables suchas the plasmid vector system chosen for the cloning and introduction ofthe recombinant DNA molecule, the plant species to be modified, theparticular structural gene, promoter elements and upstream elementsused. Persons skilled in the art are able to select and use appropriatealternatives to achieve functionality. Culture conditions for expressingdesired structural genes and cultured cells are known in the art. Alsoas known in the art, a number of both monocotyledonous anddicotyledonous plant species are transformable and regenerable such thatwhole plants containing and expressing desired genes under regulatorycontrol of the promoter molecules and upstream elements of the inventionmay be obtained. As is known to those of skill in the art, expression intransformed plants may be tissue specific and/or specific to certaindevelopmental stages or environmental influences. Truncated promoterselection and structural gene selection are other parameters which maybe optimized to achieve desired plant expression as is known to those ofskill in the art and taught herein.

The nucleotide sequences of the invention can be introduced into anyplant. The genes to be introduced can be conveniently used in expressioncassettes for introduction and expression in any plant of interest.

Such expression cassettes will comprise the transcriptional initiationregion of the invention linked to a nucleotide sequence of interest.Such an expression cassette is provided with a plurality of restrictionsites for insertion of the gene of interest to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The transcriptional cassette will include in the 5'-3' direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau et al., (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. 1989) Nucleic Acids Res.17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

The genes of the invention are provided in expression cassettes forexpression in the plant of interest. The cassette will include 5' and 3'regulatory sequences operably linked to the gene of interest. Thecassette may additionally contain at least one additional gene to becotransformed into the organism. Alternatively, the additional gene(s)can be provided on another expression cassette. Where appropriate, thegene(s) may be optimized for increased expression in the transformedplant. That is, the genes can be synthesized using plant preferredcodons for improved expression. Methods are available in the art forsynthesizing plant preferred genes. See, for example, U.S. Pat. Nos.5,380,831, 5,436, 391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequenceswhich may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The selection of an appropriate expression vector will depend upon themethod of introducing the expression vector into host cells. Typicallyan expression vector contains (1) prokaryotic DNA elements coding for abacterial replication origin and an antibiotic resistance gene toprovide for the amplification and selection of the expression vector ina bacterial host; (2) DNA elements that control initiation oftranscription such as a promoter; (3) DNA elements that control theprocessing of transcripts such as introns, transcriptiontermination/polyadenylation sequence; and (4) a reporter gene that isoperatively linked to the DNA elements to control transcriptioninitiation. Useful reporter genes include β-glucuronidase,β-galactosidase, chloramphenicol acetyl transferase, luciferase, greenfluorescent protein (GFP) and the like. Preferably the reporter gene iseither β-glucuronidase (GUS), GFP or luciferase. The generaldescriptions of plant expression vectors and reporter genes can be foundin Gruber, et al., "Vectors for Plant Transformation, in Methods inPlant Molecular Biology & Biotechnology" in Glich et al., (Eds. pp.89-119, CRC Press, 1993). Moreover GUS expression vectors and GUS genecassettes are available from Clonetech Laboratories, Inc., Palo Alto,Calif. while luciferase expression vectors and luciferase gene cassettesare available from Promega Corp. (Madison, Wis.).

Expression vectors containing genomic or synthetic fragments can beintroduced into protoplasts or into intact tissues or isolated cells.Preferably expression vectors are introduced into intact tissue. Generalmethods of culturing plant tissues are provided for example by Maki etal. "Procedures for Introducing Foreign DNA into Plants" in Methods inPlant Molecular Biology & Biotechnology, Glich et al. (Eds. pp. 67-88CRC Press, 1993); and by Phillips et al. "Cell-Tissue Culture andIn-Vitro Manipulation" in Corn & Corn Improvement, 3rd Edition Spragueet al. (Eds. pp. 345-387) American Society of Agronomy Inc. et al. 1988.

Methods of Introducing expression vectors into plant tissue include thedirect infection or co-cultivation of plant cell with Agrobacteriumtumefaciens, Horsch et al., Science, 227:1229 (1985). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer provided by Gruber, et al. supra.

Preferably, expression vectors are introduced into maize or other planttissues using a direct gene transfer method such asmicroprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using the microprojectile media delivery with the biolisticdevice. See, for example, Tomes et al. "Direct DNA transfer into intactplant cells via microprojectile bombardment" In: Gamborg and Phillips(Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods,Springer-Verlag, Berlin (1995).

The vectors of the invention can not only be used for expression ofstructural genes but may also be used in exon-trap cloning, or promotertrap procedures to detect differential gene expression in varieties oftissues, K. Lindsey et al., 1993 "Tagging Genomic Sequences That DirectTransgene Expression by Activation of a Promoter Trap in Plants",Transgenic Research 2:33-47. D. Auch & Reth, et al., "Exon Trap Cloning:Using PCR to Rapidly Detect and Clone Exons from Genomic DNA Fragments",Nucleic Acids Research, Vol. 18, No. 22, p. 6743.

This inventive promoter is based in part on the discovery that a GC rich"TATA to start" region in a plant promoter acts as a very strongnontissue specific core promoter inducing constitutive expression inplant cells. The TATA element of plant promoters of polI genes generallyhave the sequence TATA(A/T)A(A/T)A, SEQ ID NO:3, whereas the consensusof the start of transcription consists of the sequence5'...TYYTCAT(A/C)AA..3'. SEQ ID NO.:3, where the A designates thestarting base for transcription. The typical plant promoter sequence isdepicted in FIG. 1.

Sequences intervening the TATA element and the start of transcriptionhave been shown to play a significant role in transcriptional activationefficiency. The TATA binding protein has been shown to interact with theminor groove of the double helix binding to the TATA motif bending ittowards the major groove side (Kim, et al. 1993, Nature, 365:512-520).It thus follows that sequences downstream of the TATA motif that impactthis finding will affect the efficiency of stable transcriptionalcomplex formation and ultimately expression. Surveys of the "TATA tostart" regions of plant promoters show a significantly higher level ofAT-rich sequences leading to the potential of minor groove compression(Yaurawj et al Biological Abstracts Vol. 47, Issue 8, Ref 144712,"Consensus Sequences for Plant Minimal Promoters" Annual Meeting of theAmerican Society of Plant Physiologists, Jul. 29-Aug. 2, 1995, PlantPhysiology 108 [2 Supp.] 1995, 114). Generally animal promoters show aGC-rich "TATA to start" sequence that leads to a major groovecompression suggesting that average plant and animal core promotertranscriptional complexes recognize and interact with a somewhatdifferent TATA to start structure with the corresponding sequencedifference. Quite surprisingly the applicant has found that a GC-richanimal type synthetic promoter works very well in plants.

While the invention is not bound by any theory, it is possible that theAT-rich TATA motif present in a GC-rich sequence may "present itself"more prominently to the TATA-binding complex by a sharp demarcation ofthe TATA motif that would interact more tightly with the TATA-bindingcomplex. This would improve the start of transcription efficiency, byshifting the equilibria of binding to a more stabilized form, whereasthe "non-bounded TATA" version, i.e. having a higher level of AT-Richsequences flanking the T ATA motif, the TATA-binding complex wouldpotentially slide or stutter 5' or 3' to the start site and effectivelyreduce the efficiency of binding ultimately reducing transcription.Little data regarding this region of plant promoters is available exceptcrude deletions and some point mutations. The obvious design of asynthetic core promoter for plant expression would include the AT-rich"TATA to start" sequence based on surveys of known supplant promoters.However, based on the "bounded" mechanism, it is postulated by themechanism of the invention that a more efficient core promoter is aresult of a TATA motif imbedded in a GC-rich sequence.

FIG. 2 depicts the Syn II Core promoter sequence, SEQ ID NO:1 of theinvention with examples of plant core promoters aligned to the majorstart of transcription. Another example of a plant promoter 35S of CaMV(SEQ ID NO:4) are shown with percent GC-rich sequences shown at theright in parentheses. The Syn II Core sequence does not show anysignificant sequence homology to sequences in the public sequencedatabases.

The synthetic Syn II Core promoter sequence shows a 64% GC-rich "TATA tostart" sequence different from the overall 40% GC-rich sequence presentin traditional plant promoters (CaMV35S for example). The naturallyoccurring and isolated UBI core promoter which potentiates very highlevels of activity in monocots usually shows a 64% GC-rich "TATA tostart" sequence more similar to animal promoters. Such examples providedthe impetus to design a high GC-rich "TATA to start" sequence forefficient transcription in opposition to the current dogma of plant corepromoters.

Thus the invention comprises a synthetic plant core promoter sequencecomprising a TATA motif and a "TATA to start" region that is 64% GC-richor greater. In a preferred embodiment, the promoter may includerestriction endonuclease target sites for ease of cloning. In the mostpreferred embodiment, the sequence is that of SEQ ID NO:1. As will beappreciated by those of skill in the art, several base transversionswithin SEQ ID NO:1 may occur which will maintain the percent GC-contentand are intended within the scope of this invention. For exampleguanines could be replaced with cytosines and vice-versa withoutaffecting the overall efficacy of the promoter, so long as the percentGC-content is maintained.

In another embodiment, the invention comprises a synthetic upstreamelement positioned 5' to any naturally occurring or synthetic promoterfor use in plants, particularly maize gene expression.

From the activity of numerous promoters, basic elements (binding sites)have been defined. These include for example AT-rich regions from heatshock promoters, and ASF-1 binding site (AS-1) elements present inoctopine synthase (OCS) and Cauliflower Mosaic Virus promoters. AS-1 isone of the better known upstream elements and its binding sequence (OCSelement) is present in many constitutive plant promoters such as theCaMV35S, A. tumefaciens, NOS and OCS wheat histone promoters. The OCSelement was first isolated as an enhancer element in the promoter of theOCS gene where it was identified as a 16-base pair palindromic sequence(Ellis et al., (1987) EMBOJ. 6:11-16), but has been reduced to itsessential features as a TGACG motif. See U.S. Pat. No. 4,990,607incorporated herein by reference. The upstream element of the inventionhas a 71% identity to the promoter enhancer clement disclosed in U.S.Pat. No. 5,023,179 to Lam et al. The two sequences are quite differentin their flanking sequences surrounding the TGACG motif, which regionshave been shown to impact the level of transcription enhancement. Thetranscriptional enhancing activity of the OCS element correlates withthe in-vitro binding of a transcriptional factor. Similar elements werealso identified in the promoter regions of six other cDNA genes involvedin opine synthesis and three plant viral promoters including the CaMV35S promoter (Bouchez et al. 1989) supra. These elements were shown tobind the OCS transcription factor in-vitro and enhance transcription inplant cells.

In tobacco a DNA binding factor, TGA I, was shown to interactspecifically with the AS-1 element either alone or in conjunction withother promoter elements. (Katagiri et al. 1989, Nature 340:727-730).This factor was also shown to be expressed in a root-preferred manner intobacco plants. Core promoters with one or two copies of the OCSupstream element tend to potentiate gene expression whereas 4 or morerepeats of this element produce more or less constitutive activityalbeit low relative to intact 35S promoters.

Thus the invention incorporates a synthetic upstream element which canbe used with the core promoter of the invention or other core promotersto enhance gene expression. The element incorporates three OCS-likemotifs and novel intervening sequences which enhance gene expression.

FIG. 3, SEQ ID NO:2 shows the complete sequence of one embodiment(RSyn7) of the synthetic upstream element which incorporates at leastthree TGACG SEQ ID NO:5 OSC-like motifs which are indicated in bold.

Sequences flanking many elements such as the TGACG SEQ ID NO:5 motifhave been shown to have profound impacts on binding affinities of DNAbinding factors and thus play as an important role as the central motifsthemselves. (Burrows et al. 1992, Plant Molecular Biology 19:665-675,Shinder et al. 1992, Plant Cell 4:1309-1319, Foster et al. 1994, FASEBJ8:192-200). The novel sequences flanking the TGACG motifs in the Rsyn7promoter have been determined and established clear enhancement oftranscriptional activity with various promoters, particularly when usedwith the Syn II Core promoter.

Rsyn7 upstream element has been cloned upstream of the Syn II Corepromoter driving a GUS construct and has yielded levels of GUS activityin transgenic maize plants approximately ten-fold higher than theubiquitin promoter, the strongest maize promoter to date.

In yet another aspect of the present invention, at least one upstreamactivating region (UAR), which is different from the synthetic upstreamelement, is operably linked to the synthetic core promoter. The UAR maybe used alone or in combination with the synthetic upstream elementdescribed herein. Preferably, the upstream activating regions of thecauliflower mosaic virus (CaMV) 35S promoter and the maize Ubi-1 genepromoter are utilized. Additionally, sequences having sequencesimilarity to these UARs may be utilized as long as such sequencesretain the ability to enhance promoter activity. Enhancement can bemeasured by assaying for levels of transcripts or alternatively proteinproduction.

CaMV 35S UARs have been well studied in the art. The complete nucleotidesequence of the CaMV circular double-strand DNA has been established inthe art. See Guilley et al. (1980) Cell 21:285-294. The 35S promotertranscribes the major 35S RNA transcript from the circular viral genomeby nucleus RNA polymerase II. See Guilley et al. (1982) Cell 30:763-773.Moreover, the 35S UARs can function with a heterologous promoter andincrease expression of a gene of interest in cells and transgenicplants. Shah et al. (1986) Science 233:478-481. Multiple cis regulatoryelements for the activity of the CaMV 35S promoter have been identified.See Odell et al. (1985) Nature 313:810-812; Fang et al. (1989) PlantCell 1:141-150.

In the present invention, a large fragment of the upstream activatingregions (UARs) of the CaMV 35S promoter can be utilized to enhance theactivity of the core synthetic promoter. The size of the UAR can, forexample, range from about 15 base pairs to about 850 base pairs,preferably from about 20 to about 500, more preferably from about 20-25to about 50-200 base pairs. A preferred region of the 35S CaMV upstreamregion includes sequences from about -421 to about -90. It is recognizedthat modifications, in length and nucleotide sequence can be made to theregion and still result in enhanced activity of the core syntheticpromoter. Such modifications can be tested for effect on activity byusing expression systems as set forth in the Experimental Section of thepresent application. The numbers on the UAR sequence diagram indicatethe position upstream from the transcription start site, or +1 positionof the 35S structural gene. For example, -25 means a position 25 basepairs upstream from the transcription start site of the 35S structuralgene.

The upstream activating region of the maize ubiquitin gene Ubi-1 canalso be utilized in the invention. The sequence of the Ubi-1 genetranscription regulatory region is disclosed in U.S. Pat. No. 5,510,474.See also Christensen et al. (1992) Plant Mol. Biol. 18:675-689; Cornejoet al. (1993) Plant Mol. Biol. 23:567-581; Takimoto et al. (1994) PlantMol. Biol. 26:1007-1012; and Christensen et al. (1996) Transgenic Res.5:213-218. The UAR of the Ubi-1 gene promoter comprises preferably fromabout -867 to about -54. As indicated above for the 35S UAR,modifications of the Ubi-1 UAR that still function to enhance theactivity of the core promoter are encompassed.

While the full sequence of the ubiquitin promoter has been published,this is the first disclosure of the Ubi UAR. Thus, the inventiondiscloses the UAR of the ubiquitin promoter as well as the Ubi UAR incombination with any promoter. Additionally, methods for using the UbiUAR to enhance activity of promoters are encompassed.

The upstream activating regions as described herein can be linked withthe synthetic core promoter and/or other upstream elements by anyconventional method that is generally known in the art as long as anoperative element or promoter is constructed. The upstream activatingregions are generally operably linked to the 5' end of the corepromoter. When the synthetic upstream clement is also present, theupstream activating regions can be linked to either the 5' end of thesynthetic upstream element, the 3' end of the core synthetic promoter orinserted between the synthetic upstream element and the synthetic corepromoter. In a preferred embodiment the upstream activating regions arelinked in close proximity to the synthetic upstream element, if present,and the synthetic core promoter. By close proximity is intended withinfrom about 1 to about 50 nucleotides. However, it is recognized thatmore than 50 nucleotides may separate the elements. The upstreamactivating regions can be in the 5' to 3' direction or the 3' to 5'direction, but preferably in the 5' to 3' direction at the 5' end of thesynthetic core promoter or the synthetic upstream element.

One or multiple copies of the upstream activating regions can be used.When multiple copies are utilized, they can be tandem repeats of one UARor combinations of several UARs. In this manner, the level of expressionof a nucleotide sequence of interest can be controlled by the number ofUARs present in the promoter construction since the results indicatethat increased expression levels are obtained with increased numbers ofUARs. Thus, the invention provides methods for regulating levels ofexpression of a gene or nucleotide sequence of interest.

As indicated, multiple copies of a UAR can be used to enhance theactivity of the operably linked promoter. As noted, multiple copies ofthe same or different UARs can be utilized. For example, any combinationof CaMV 35S UARs and maize Ubi-1 gene UARs can be utilized.

The promoters of this invention having one or more UARs as describedabove can be provided in expression cassettes and such cassettscontained in plasmid or viral vectors. Such vectors can be used fortransformation of bacteria and plant cells. Transgenic plants can beultimately regenerated from such transformed plant cells.

The UARs incorporated into plant promoters can substantially enhancetranscription activity in transgenic plants. For example, one or morecopies of the upstream activating region of the maize Ubi-1 gene can beoperably linked to a promoter having the core synthetic promotersequence and the synthetic upstream element of the invention. Thepromoter constructs of the invention can be operably linked to anynucleotide sequence or gene of interest. The promoter construct, forexample, can be used to enhance oxalate oxidase gene expression intransgenic plants. Oxalate oxidase is a plant enzyme implicated in plantdefense mechanisms against pathogens attack. The enzyme degrades thechemical compound oxalic acid secreted by plant pathogens. See e.g., PCTPublication No. WO 92/14824. Increasing the oxalate oxidase level inplants such as sunflower will lead to increased plant resistance toplant pathogens.

The following examples are for illustration purposes only and areintended in no way to limit the scope or application of the presentinvention. Those of skill in the art will appreciate that manypermutations can be achieved and are in fact intended to be within thescope of the invention. All reference citations throughout thespecification are expressly hereby incorporated by reference.

EXAMPLE 1

Plasmids were designed using the multiple cloning site ofpBlueScriptIIKS+ from Stratagene. (To facilitate cloning of thedifferent combination of elements). Oligonucleotides containing thesequences of the elements were synthesized with restriction endonucleasesites at the ends. Thus elements could be added or removed and replacedas needed. GUS and Luciferase were used for reporter genes.

For transient assays, plasmid DNA was introduced into intact 3-day-oldmaize seedlings by particle bombardment. Following 16 hours incubationat 25EC in the dark, expression was assayed by measuring GUS enzymeactivity in root and shoot extracts from each seedling to determine ifany tissue-preferred expression was demonstrated. GUS activity wasmeasured using a GUS-Light assay kit from Tropix (47 Wiggins Avenue,Bedford, Mass. 01730).

Constructs that gave high levels of expression were introduced into acell line to produce stable transformants. These stable transformants(TO) were assayed by PCR to determine the presence of the GUS gene byMUG (4-methylumelliferyl-glucuronide) assay to quantify the activitylevel of the GUS protein being produced. When the plants were ready tobe transferred to the greenhouse they were assayed histochemically withX-gluc to determine where the GUS product was being synthesized. Plantsdemonstrating preferred expression levels were grown in the greenhouseto V6 stage.

EXAMPLE 2

Construction of Plasmids Containing the Syn II Core Promoter.

Standard molecular biological techniques were carried out according toManiantis et al. (1982) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. All plasmids utilizedin the invention can be prepared according to the directions of thespecification by a person of ordinary skill in the art without undueexperimentation employing materials readily available in the art.

Oligos N306 SEQ ID NO:6 5'-TCGACACTGC AGCTCTAGGG ATGGTAGCGC AGGGTGCGTAGGTACGTATT TATAGCCGCT CGAGTG-3' and N307 SEQ ID NO:75'-GATCCACTCGAGCGGCTATA AATACGTACC TACGCACCCT GCGCTACCAT CCTAGAGCT GCAGTG-3' weresynthesized according to directions on an automated DNA synthesizer(such as Applied Biosystems Inc. DNA Synthesizer (Model 380B). Theseautomated synthesizers are commercially available. The oligos were thenligated to the BamHI fragment of the pBlueScriptIlKS+ plasmid comprisingof the β-glucuronidase gene interrupted by the maize ADH1 intron 1region. A map of a plasmid incorporating both the Syn II Core promoterand the upstream element is disclosed as FIG. 4. Several otherembodiments are shown in other plasmids depicted in FIG. 5. Plasmidnumbers are shown to the right of each promoter diagram with thecorresponding legend placed below the diagrams. The top diagram showsthe complete transplant transcriptional unit with the subsequentdiagrams focusing on the salient differences between 35S and Syn II Corepromoters. The legend shows the number and nature of the variouspromoter subelements, the sequence if relatively short, the source ofthe element and position relative to the start of transcription.

The sequence of the core promoter consists of 35 base pairs with enzymesites upstream of a TATA box and a start of transcription with 10 to 15base pairs downstream. Upstream elements (Gal 4-binding sites, Rsyn,AT-GBL etc.) were fused to the core sequence with ADHI-intron anddifferent marker genes (LUC or GUS) and were demonstrated functionalboth in transient assays (FIG. 6) and Rsyn stably transformed plants(FIG. 7).

EXAMPLE 3

Construction of Upstream Element Rsyn7 Fused to Syn II Core PromoterResulting in Plasmids PHP5903 and PHP6086.

Oligos for constructing the Rsyn7 promoter subelement N1965: (SEQ IDNO:8) GATCCTATGA CGTATGGTAT GACGTGTGTT CAAGATGATG ACTTCAAACC TACCTATGACGTATGGTATG ACGTGTGTCG ACTGATGACT TA-3' and N 1966: (SEQ ID NO:9)GATCTAAGTC ATCAGTCGAC ACACGTCATA CCATACGTCA TAGGTAGGTT TGAAGTCATCATCTTGAACA CACGTCATAC CATACGTCA TAG-3' were synthesized as earlierdescribed. The oligos were annealed and cloned into a PHP3398 plasmidupstream of the Syn II Core sequence and resulted in several versions ofthe original Rsyn7 sequence due to spontaneous deletions. The Rsyn7-2version involved a single base deletion resulting in a 3× reiterativeTGACG motif upstream of the Syn II Core promoter (Rsyn7::LUC, P5903).The LUC coding sequence was replaced by GUS coding sequence to producethe Rsyn7::GUS construct P6086. P6086 was later introduced intotransgenic maize resulting in high levels of constitutive activity infour of the six active events examined (FIG. 7).

The progeny from T0 plants from several transformation events wereexamined and GUS activity ranging from 1 to 400 PPM (micrograms GUSenzyme/GFW in root tissue of a 7-day old seedlings) FIG. 8. These T0 andT1 plants generally produced 4X-10 X greater GUS activity than plantsharboring the ubiquitin::GUS reporter gene.

Thus from the foregoing, it can be seen that the invention accomplishesat least all of its objectives.

EXAMPLE 4

Transformation and Expression with Syn II Core Promoter and/or Rsyn7Upstream Element.

Using transient bombardment assays the Syn II Core promoter sequence wascompared against the 35S core sequence either alone or in conjunctionwith numerous activation elements. FIG. 6 is a depiction of transientassay data using the plasmids incorporating the promoter sequences ofthe invention and shows transient GUS or LUC activity in three-day oldmaize roots or BMS callus bombarded with chimeric promoter::GUS or LUCconstructs. The -33 CaMV35S in the Syn II Core promoter versions of thesynthetic promoter::GUS (or LUC) constructs were bombarded intothree-day old roots (or cultured BMS calli as described hereinafter) andassayed for enzyme activity 20 hours after bombardments. The data shownare the raw enzyme units of a compilation of at least three experimentsand have not been normalized in any fashion due to the inherentvariability of the transient assays. Control plasmids 1654 and 3537 arethe LUC constructs tested in maize BMS calli. There is approximately 4to 20 fold difference in transient activity between the 35S and Syn IICore versions. The Y axis is in log scale. Both core promoters weredriving a GUS containing construct (FIGS. 4 and 5) and generated a basallevel of activity (FIG. 6). However when activator elements were placedupstream of the TATA motif, the Syn II Core provided generally higherlevels of activity (2-4 fold better) in corn cells than when theactivator elements were placed upstream of the 35S core (FIG. 6).

The Syn II Core sequence has been shown to enhance activity in stablytransformed plants. Further with certain activator sequences upstream ofthe TATA element activity levels in stably transformed corn plantsreached levels ten-fold greater than maize ubiquitin constructs whichproduces extremely high levels of activity.

FIGS. 7 and 8 show GUS activity levels from isolated tissues of VT stageT0 plants and root tissue from T1 seedlings, respectively. These datademonstrate that this core sequence can participate in potentiating veryhigh levels of activity as a functional partner for the active chimericpromoters. FIG. 7 shows the Rsyn7::GUS (6086) activity in T0 maizeplants. VT stage plants with ears post pollinated 3 to 8 days weredissected and assayed for GUS activity. 7A depicts GUS expression indesignated tissues. 7B depicts a schematic of a corn plant with sites ofmeasurement indicated. Plants from ro events that demonstrated a rangeof activities with the Rsyn7 promoter were assayed. Log scale againnoted. The activity range for UBI::GUS plants is indicated at the rightof graph for comparisons. These data demonstrate that the Rsyn7 promotercan increase activity to ten-fold above levels of the ubiquitin promoteryet shows little tissue preference making the Rsyn7 suitable as a strongconstitutive promoter.

FIG. 8 depicts GUS activity in root segments of a segregating populationof maize T1 transgenic seedlings containing the Rsyn7::GUS (6086) or theUBI::GUS (3953) construct. 1 cm root segments from six to seven-day oldtransgenic maize seedlings were dissected, weighed and assayed for GUSusing GUS-light kit. Activity is represented as parts per million offresh weight. The root activity of several T1 plants harboring theRsyn7::GUS promoter shows higher activity than much of the activitylevels produced by the UBI promoter. This is consistent with data fromT0 transgenic plant. Activity levels in Rsyn7::GUS containing youngleaves are also much higher than the activity levels ofUBI::GUS-containing young leaves (data not shown). The Syn II Coresequence was shown to function well with a variety of upstream elementsincluding GAL 4 binding sites, Rsyn7 elements, GBL elements, etc.

FIG. 9 shows GUS expression of three synthetic promoters in T0transgenic maize plants. Dissected tissues (See FIG. 7B) from VT stagetransgenic T0 plants harboring Rsyn7 (Rsyn), Atsyn or the Syn II Corealone (syn-core) promoter::GUS constructs were quantitatively assayedfor GUS activity. Each circle represents an average of tissue activityof transgenic maize plants from a single transformation event. TheTGACG-motif corresponds to the Rsyn7 sequence and the "AT-com" motifrefers to the consensus AT-like composite element, Atcom, from W.Gurley, et al. 1993. In: Control of Plant Gene Expression. ed. by DeshPal Verma. CRC press, Boca Raton, Fla. pp. 103-123. Syn-core refers tothe Syn II Core promoter sequence containing the TATA element and thestart of transcription.

EXAMPLE 5

Construction of Rsyn7 Promoter Having the Upstream Activating Regionsfrom the CaMV 35S Gene and the Maize Ubi-1 Gene.

To construct an expression vector having 35SU (upstream activatingregions from 35S gene)-Rsyn7 promoter, PHP413 was digested with BglIIand EcoRV. The staggered/sticky ends of the linearized vector werefilled in by Klenow in the presence of dNTP. The 2× CaMV fragment wasblunt end ligated into BamH1 digested PHP6086 after filling the BamHIends. The CaMV 35S-Rsyn7 fragment was then cut out from the newconstruct by digestion with Xba1 and Pst 1, and ligated into the 4 kbXbaI-Pst1 vector from PHP9925 to form the expression vector of35SU-Rsyn7::GUS (PHP9778).

To construct an expression vector having UbiU (upstream activatingelements from the maize Ubi-1 gene)-Rsyn7 promoter, the Xba1-Spe1fragment from PHP8277 was ligated into the Xba1 site of PHP6086 to formPHP10539, into the Xba1 site of PHP10970 to form PHP10971, and into theXba1 site of PHP10971 to form the expression vector of Ubi-1-Rsyn7::GUS(PHP 10972).

Those sequences not referenced otherwise include:

SEQ ID NO: 11 sets forth the 35S UAR.

SEQ ID NO: 12 sets forth the SCPI promoter sequence, (35S UAR operablylinked to core promoter of SEQ ID NO: 1).

SEQ ID NO: 13 sets forth the Ubi1 UAR.

SEQ ID NO: 14 sets forth SCP1 operably linked to the oxalate oxidasecoding sequence operably linked with the PinII terminator.

SEQ ID NO: 16 sets forth the UCP2 promoter sequence (2 copies of Ubi1UAR operably with the core promoter).

SEQ ID NO: 18 set forth the UCP4 promoter sequence (4 copies of Ubi1 UARoperably with the core promoter).

EXAMPLE 6

Transformation and Expression of promoter constructs.

The various promoters::GUS fragments were cloned into a Bin9 binaryvector that contains ALS3::NPTII as selection marker for generatingtransgenic sunflower callus or Arabidopsis.

For transient expression, SMF3 sunflower seeds were planted ingreenhouse. 15-day-old seeds after pollination were collected from theplants and used in the transient expression system. After removing thepericarp, the cotyledons with seed coats were sterilized by incubationin 20% bleach at RT for 15 minutes, and washed four times with steriledouble distilled water. The cotyledons were then incubated on 3MM filterwetted with MS medium overnight before they were bombarded according tothe method disclosed by Klein et al. (1989) Proc. Natl. Acad Sci. USA86:6681-6685, hereby incorporated by reference thereto. GUS activity wasanalyzed 20 hours after bombardment using the GUS-Light assay kit fromTropix according to the manufacturer's protocol.

For leaf disc transformation, young expanded SMF3 sunflower leaf from30-day-old sunflower was harvested and sterilized in 20% bleach with acouple of drops of Tween 20 for 20 minutes. Leaf discs were preparedfrom the sterile leaves after washing them with sterile double distilledwater 4 times. The leaf discs were then incubated for 10 minutes ininoculation medium (12.5 mM MES, 1 g/l NH₄ Cl, and 0.3 g/l MgSO₄)containing Agrobacterium (EHA105) transformed with the vector constructsto be tested at A600=0.75. The leaf discs were then grown for 3 days innon-selection medium and were then transferred to selection medium.

To transform Arabidopsis, Arabidopsis were grown in greenhouse to thestage when bolts start to emerge at 15 plants/pot. The emerging boltswere clipped off to encourage the growth of multiple secondary bolts.After 7 days, the plants were ready for infiltration. Agrobacterium(EHA105) carrying the construct to be tested was cultured at 28 ° C. towhen A600 was between 0.65 and 0.8. The cells were harvested ininoculation medium (4.3 g/l of MS salt, 0.5 mg/l of nicotinic acid, 0.5mg/l of pyridoxine-HCl, 1 mg/l of Thiamine-HCl, 0.1 g of myo-inositol, 1g/l of casamino acids, 0.01 mg of BAP, 68.5 g/l of sucrose, and 36 g/lof glucose) at A600 of 7.5.

The clipped plants to be transformed were inverted into a 250 ml beakercontaining the above Agrobacterium solution. The beaker was placed intoa bell jar and was vacuumed until bubbles formed on leaf and stemsurface. After 15 minutes of infiltration, the vacuum was released andthe plants were removed from the beaker, laid on its side in a plasticflat, and covered with plastic wrap. The plants were set upright andgrown in greenhouse for four weeks before seeds were harvested.Transgenic seeds were selected by planting the seeds on a medium platecontaining 65 μg/ml of kanamycin.

In transient expression assays, the Rsyn7 promoter in PHP10464 has 15%of the 35S promoter (PHP9925) activity, whereas the 35SU-Rsyn7 promoter(PHP9778) (hereinafter SCP1 promoter) has about 107% of the 35S promoteractivity (FIG. 10). Thus, the upstream activating region of the CaMV 35Sgene increased the Rsyn7 activity by about 6 fold.

The maize Ubi-1 upstream element (UbiU) has similar effects on the Rsyn7promoter in transient assays. When the upstream activating region (UAR)of the maize Ubi-1 was fused to Rsyn7, the GUS enzyme activity increasedwith the number of copy of the UAR. In the presence of three copies ofthe UbiU, GUS activity increased by about 4-fold. This additive effectof UbiU was not observed when placed in the context of the maizeubiquitin promoter (PHP 11974). This suggests that replacement of themaize Ubi1 core promoter with Rsyn7 may convert the monocot Ubi1promoter into a highly active promoter in dicot plants (FIG. 11).

In the stably transformed sunflower callus, GUS expression is 20% higherbehind the control of the 35SU-Rsyn7 (SCP1 promoter) promoter than whenbehind the control of the 35S CaMV promoter(FIG. 12).

The results of the transgenic callus assay are given in FIG. 13. TheRsyn7 promoter containing a single copy of UbiU (PHP10991) (hereinafterUCP1 promoter) (SEQ ID NO:15) exhibited promoter activity of about 3times that of the 35SU-Rsyn7 (SCP1) promoter (PHP 10940). Three copiesof UbiU (PHP 10993) increased Rsyn7 promoter activity to about 7 timesthat of the 35SU-Rsyn7 promoter. The 3×UbiU-Rsyn7 (UCP3 promoter) (SEQID NO: 17) is by far the strongest promoter in sunflower tissues.

To determine the activity and tissue-specificity of the enhanced Rsyn7promoters, stably-transformed sunflower and Arabidopsis were generatedthrough Agrobacterium-mediated transformation. The histochemicalstaining of GUS expression in transgenic T1 Arabidopsis indicates that35SU-Rsyn7 (SCP1 promoter) (PHP10940) has identical tissue-specificityand similar activity as 35S CaMV promoter (PHP10989). Both promotersexpress GUS in leaf stem, petiole, and floral parts. UbiU-Rsyn7 (USCP1promoter) (PHP10991) exhibits higher activity than maize Ubi-1 promoter(PHP 11031) in Arabidopsis stem and leaf tissues.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 18                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 72 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GGATCCACTC GAGCGGCTAT AAATACGTAC CTACGCACGC TGCGCTACCA TC - #CCGAGCAC         60                                                                          #       72                                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 96 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic nucleic acid"/desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - GGATCCTATG CGTATGGTAT GACGTGTGTT CAAGATGATG ACTTCAAACC TA - #CCTATGAC         60                                                                          #       96         GTCG ACTGATGACT TAGATC                                     - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #  18              AA                                                         - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #    40            TTCA TTTCATTTGG AGAGGAAACG                                 - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 5 base p - #airs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #             5                                                               - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic oligonucleotide"sc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - TCGACACTGC AGCTCTAGGG ATGGTAGCGC AGGGTGCGTA GGTACGTATT TA - #TAGCCGCT         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic oligonucleotide"sc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - GATCCACTCG AGCGGCTATA AATACGTACC TACGCACCCT GCGCTACCAT CC - #CTAGAGCT         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 92 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic oligonucleotide"sc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - GATCCTATGA CGTATGGTAT GACGTGTGTT CAAGATGATG ACTTCAAACC TA - #CCTATGAC         60                                                                          #          92      GTCG ACTGATGACT TA                                         - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 92 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic oligonucleotide"sc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - GATCTAAGTC ATCAGTCGAC ACACGTCATA CCATACGTCA TAGGTAGGTT TG - #AAGTCATC         60                                                                          #          92      ATAC CATACGTCAT AG                                         - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 72 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic nucleic acid corec                                              #with G/C transversions"                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - GGATCCACTC GAGCGGCTAT AAATASSTAS STASSSASSS TSSSSTASSA TC - #CCGAGCAC         60                                                                          #       72                                                                    - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 332 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - CGTCAACATG GTGGAGCACG ACACTCTCGT CTACTCCAAG AATATCAAAG AT - #ACAGTCTC         60                                                                          - AGAAGACCAA AGGGCTATTG AGACTTTTCA ACAAAGGGTA ATATCGGGAA AC - #CTCCTCGG        120                                                                          - ATTCCATTGC CCAGCTATCT GTCACTTCAT CAAAAGGACA GTAGAAAAGG AA - #GGTGGCAC        180                                                                          - CTACAAATGC CATCATTGCG ATAAAGGAAA GGCTATCGTT CAAGATGCCT CT - #GCCGACAG        240                                                                          - TGGTCCCAAA GATGGACCCC CACCCACGAG GAGCATCGTG GAAAAAGAAG AC - #GTTCCAAC        300                                                                          #         332      GTGG ATTGATGTGA TG                                         - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 499 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - CGTCAACATG GTGGAGCACG ACACTCTCGT CTACTCCAAG AATATCAAAG AT - #ACAGTCTC         60                                                                          - AGAAGACCAA AGGGCTATTG AGACTTTTCA ACAAAGGGTA ATATCGGGAA AC - #CTCCTCGG        120                                                                          - ATTCCATTGC CCAGCTATCT GTCACTTCAT CAAAAGGACA GTAGAAAAGG AA - #GGTGGCAC        180                                                                          - CTACAAATGC CATCATTGCG ATAAAGGAAA GGCTATCGTT CAAGATGCCT CT - #GCCGACAG        240                                                                          - TGGTCCCAAA GATGGACCCC CACCCACGAG GAGCATCGTG GAAAAAGAAG AC - #GTTCCAAC        300                                                                          - CACGTCTTCA AAGCAAGTGG ATTGATGTGA TGATCCTATG CGTATGGTAT GA - #CGTGTGTT        360                                                                          - CAAGATGATG ACTTCAAACC TACCTATGAC GTATGGTATG ACGTGTGTCG AC - #TGATGACT        420                                                                          - TAGATCCACT CGAGCGGCTA TAAATACGTA CCTACGCACC CTGCGCTACC AT - #CCCTAGAG        480                                                                          #499               ACA                                                        - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 813 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Zea mays                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - TCTAGAGATA ATGAGCATTG CATGTCTAAG TTATAAAAAA TTACCACATA TT - #TTTTTTGT         60                                                                          - CACACTTGTT TGAAGTGCAG TTTATCTATC TTTATACATA TATTTAAACT TT - #ACTCTACG        120                                                                          - AATAATATAA TCTATAGTAC TACAATAATA TCAGTGTTTT AGAGAATCAT AT - #AAATGAAC        180                                                                          - AGTTAGACAT GGTCTAAAGG ACAATTGAGT ATTTTGACAA CAGGACTCTA CA - #GTTTTATC        240                                                                          - TTTTTAGTGT GCATGTGTTC TCCTTTTTTT TTGCAAATAG CTTCACCTAT AT - #AATACTTC        300                                                                          - ATCCATTTTA TTAGTACATC CATTTAGGGT TTAGGGTTAA TGGTTTTTAT AG - #ACTAATTT        360                                                                          - TTTTAGTACA TCTATTTTAT TCTATTTTAG CCTCTAAATT AAGAAAACTA AA - #ACTCTATT        420                                                                          - TTAGTTTTTT TATTTAATAA TTTAGATATA AAATAGAATA AAATAAAGTG AC - #TAAAAATT        480                                                                          - AAACAAATAC CCTTTAAGAA ATTAAAAAAA CTAAGGAAAC ATTTTTCTTG TT - #TCGAGTAG        540                                                                          - ATAATGCCAG CCTGTTAAAC GCCGTCGACG AGTCTAACGG ACACCAACCA GC - #GAACCAGC        600                                                                          - AGCGTCGCGT CGGGCCAAGC GAAGCAGACG GCACGGCATC TCTGTCGCTG CC - #TCTGGACC        660                                                                          - CCTCTCGAGA GTTCCGCTCC ACCGTTGGAC TTGCTCCGCT GTCGGCATCC AG - #AAATTGCG        720                                                                          - TGGCGGAGCG GCAGACGTGA GCCGGCACGG CAGGCGGCCT CCTCCTCCTC TC - #ACGGCACG        780                                                                          #        813       CCTT TCCCACCGCT CCT                                        - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1600 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - GATCTGAGTC TAGAAATCCG TCAACATGGT GGAGCACGAC ACTCTCGTCT AC - #TCCAAGAA         60                                                                          - TATCAAAGAT ACAGTCTCAG AAGACCAAAG GGCTATTGAG ACTTTTCAAC AA - #AGGGTAAT        120                                                                          - ATCGGGAAAC CTCCTCGGAT TCCATTGCCC AGCTATCTGT CACTTCATCA AA - #AGGACAGT        180                                                                          - AGAAAAGGAA GGTGGCACCT ACAAATGCCA TCATTGCGAT AAAGGAAAGG CT - #ATCGTTCA        240                                                                          - AGATGCCTCT GCCGACAGTG GTCCCAAAGA TGGACCCCCA CCCACGAGGA GC - #ATCGTGGA        300                                                                          - AAAAGAAGAC GTTCCAACCA CGTCTTCAAA GCAAGTGGAT TGATGTGATG AT - #CCTATGCG        360                                                                          - TATGGTATGA CGTGTGTTCA AGATGATGAC TTCAAACCTA CCTATGACGT AT - #GGTATGAA        420                                                                          - CGTGTGTCGA CTGATGACTT AGATCCACTC GAGCGGCTAT AAATACGTAC CT - #ACGCACCC        480                                                                          - TGCGCTACCA TCCCTAGAGC TGCAGCTTAT TTTTACAACA ATTACCAACA AC - #AACAAACA        540                                                                          - ACAAACAACA TTACAATTAC TATTTACAAT TACAGTCGAC CCGGGATCCA TG - #GGGTACTC        600                                                                          - CAAAACCCTA GTAGCTGGCC TGTTCGCAAT GCTGTTACTA GCTCCGGCCG TC - #TTGGCCAC        660                                                                          - CGACCCAGAC CCTCTCCAGG ACTTCTGTGT CGCCGACCTC GACGGCAAGG CG - #GTCTCGGT        720                                                                          - GAACGGGCAC ACGTGCAAGC CCATGTCGGA GGCCGGCGAC GACTTCCTCT TC - #TCGTCCAA        780                                                                          - GTTGGCCAAG GCCGGCAACA CGTCCACCCC GAACGGCTCC GCCGTGACGG AG - #CTCGACGT        840                                                                          - GGCCGAGTGG CCCGGTACCA ACAAGCTGGG TGGTGTCATG AACCGCGTGG AT - #TTTGGTCC        900                                                                          - CGGAGGGACC AACCCACCAC ACATCCACCC GCGTGCCACC GAGATCGGCA TC - #GTGATGAA        960                                                                          - AGGTGAGCTT CTCGTGGGAA TCCTTGGCAG CCTCGACTCC GGGAACAAGC TC - #TACTCGAG       1020                                                                          - GGTGGTGCGC GCCGGAGAGA CGTTCCTCAT CCCACGGGGC CTCATGCACT TC - #CAGTTCAA       1080                                                                          - CGTCGGTAAG ACCGAGGCCT CCATGGTCGT CTCCTTCAAC AGCCAGAACC CC - #GGCATTGT       1140                                                                          - CTTCGTGCCC CTCACGCTCT TCGGCTCCAA CCCGCCCATC CCAACGCCGG TG - #CTCACCAA       1200                                                                          - GGCACTCCGG GTGGAGGCCA GGGTCGTGGA ACTTCTCAAG TCCAAGTTTG CC - #GCTGGGTT       1260                                                                          - TTAATTTCTA GGATCCTCTA GAGTCGAACC TAGACTTGTC CATCTTCTGG AT - #TGGCCAAC       1320                                                                          - TTAATTAATG TATGAAATAA AAGGATGCAC ACATAGTGAC ATGCTAATCA CT - #ATAATGTG       1380                                                                          - GGCATCAAAG TTGTGTGTTA TGTGTAATTA CTAGTTATCT GAATAAAAGA GA - #AAGAGATC       1440                                                                          - ATCCATATTT CTTATCCTAA ATGAATGTCA CGTGTCTTTA TAATTCTTTG AT - #GAACCAGA       1500                                                                          - TGCATTTCAT TAACCAAATC CATATACATA TAAATATTAA TCATATATAA TT - #AATATCAA       1560                                                                          #  1600            AATC TAGTCTAGGT GTGTTTTGCC                                 - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 994 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - TCTAGAGATA ATGAGCATTG CATGTCTAAG TTATAAAAAA TTACCACATA TT - #TTTTTTGT         60                                                                          - CACACTTGTT TGAAGTGCAG TTTATCTATC TTTATACATA TATTTAAACT TT - #ACTCTACG        120                                                                          - AATAATATAA TCTATAGTAC TACAATAATA TCAGTGTTTT AGAGAATCAT AT - #AAATGAAC        180                                                                          - AGTTAGACAT GGTCTAAAGG ACAATTGAGT ATTTTGACAA CAGGACTCTA CA - #GTTTTATC        240                                                                          - TTTTTAGTGT GCATGTGTTC TCCTTTTTTT TTGCAAATAG CTTCACCTAT AT - #AATACTTC        300                                                                          - ATCCATTTTA TTAGTACATC CATTTAGGGT TTAGGGTTAA TGGTTTTTAT AG - #ACTAATTT        360                                                                          - TTTTAGTACA TCTATTTTAT TCTATTTTAG CCTCTAAATT AAGAAAACTA AA - #ACTCTATT        420                                                                          - TTAGTTTTTT TATTTAATAA TTTAGATATA AAATAGAATA AAATAAAGTG AC - #TAAAAATT        480                                                                          - AAACAAATAC CCTTTAAGAA ATTAAAAAAA CTAAGGAAAC ATTTTTCTTG TT - #TCGAGTAG        540                                                                          - ATAATGCCAG CCTGTTAAAC GCCGTCGACG AGTCTAACGG ACACCAACCA GC - #GAACCAGC        600                                                                          - AGCGTCGCGT CGGGCCAAGC GAAGCAGACG GCACGGCATC TCTGTCGCTG CC - #TCTGGACC        660                                                                          - CCTCTCGAGA GTTCCGCTCC ACCGTTGGAC TTGCTCCGCT GTCGGCATCC AG - #AAATTGCG        720                                                                          - TGGCGGAGCG GCAGACGTGA GCCGGCACGG CAGGCGGCCT CCTCCTCCTC TC - #ACGGCACG        780                                                                          - GCAGCTACGG GGGATTCCTT TCCCACCGCT CCTACTAGAA CTAGTGGATC CT - #ATGCGTAT        840                                                                          - GGTATGACGT GTGTTCAAGA TGATGACTTC AAACCTACCT ATGACGTATG GT - #ATGACGTG        900                                                                          - TGTCGACTGA TGACTTAGAT CCACTCGAGC GGCTATAAAT ACGTACCTAC GC - #ACCCTGCG        960                                                                          #       994        TGCA TGCTTATTTT TACA                                       - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1807 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - TCTAGAGATA ATGAGCATTG CATGTCTAAG TTATAAAAAA TTACCACATA TT - #TTTTTTGT         60                                                                          - CACACTTGTT TGAAGTGCAG TTTATCTATC TTTATACATA TATTTAAACT TT - #ACTCTACG        120                                                                          - AATAATATAA TCTATAGTAC TACAATAATA TCAGTGTTTT AGAGAATCAT AT - #AAATGAAC        180                                                                          - AGTTAGACAT GGTCTAAAGG ACAATTGAGT ATTTTGACAA CAGGACTCTA CA - #GTTTTATC        240                                                                          - TTTTTAGTGT GCATGTGTTC TCCTTTTTTT TTGCAAATAG CTTCACCTAT AT - #AATACTTC        300                                                                          - ATCCATTTTA TTAGTACATC CATTTAGGGT TTAGGGTTAA TGGTTTTTAT AG - #ACTAATTT        360                                                                          - TTTTAGTACA TCTATTTTAT TCTATTTTAG CCTCTAAATT AAGAAAACTA AA - #ACTCTATT        420                                                                          - TTAGTTTTTT TATTTAATAA TTTAGATATA AAATAGAATA AAATAAAGTG AC - #TAAAAATT        480                                                                          - AAACAAATAC CCTTTAAGAA ATTAAAAAAA CTAAGGAAAC ATTTTTCTTG TT - #TCGAGTAG        540                                                                          - ATAATGCCAG CCTGTTAAAC GCCGTCGACG AGTCTAACGG ACACCAACCA GC - #GAACCAGC        600                                                                          - AGCGTCGCGT CGGGCCAAGC GAAGCAGACG GCACGGCATC TCTGTCGCTG CC - #TCTGGACC        660                                                                          - CCTCTCGAGA GTTCCGCTCC ACCGTTGGAC TTGCTCCGCT GTCGGCATCC AG - #AAATTGCG        720                                                                          - TGGCGGAGCG GCAGACGTGA GCCGGCACGG CAGGCGGCCT CCTCCTCCTC TC - #ACGGCACG        780                                                                          - GCAGCTACGG GGGATTCCTT TCCCACCGCT CCTACTAGAG ATAATGAGCA TT - #GCATGTCT        840                                                                          - AAGTTATAAA AAATTACCAC ATATTTTTTT TGTCACACTT GTTTGAAGTG CA - #GTTTATCT        900                                                                          - ATCTTTATAC ATATATTTAA ACTTTACTCT ACGAATAATA TAATCTATAG TA - #CTACAATA        960                                                                          - ATATCAGTGT TTTAGAGAAT CATATAAATG AACAGTTAGA CATGGTCTAA AG - #GACAATTG       1020                                                                          - AGTATTTTGA CAACAGGACT CTACAGTTTT ATCTTTTTAG TGTGCATGTG TT - #CTCCTTTT       1080                                                                          - TTTTTGCAAA TAGCTTCACC TATATAATAC TTCATCCATT TTATTAGTAC AT - #CCATTTAG       1140                                                                          - GGTTTAGGGT TAATGGTTTT TATAGACTAA TTTTTTTAGT ACATCTATTT TA - #TTCTATTT       1200                                                                          - TAGCCTCTAA ATTAAGAAAA CTAAAACTCT ATTTTAGTTT TTTTATTTAA TA - #ATTTAGAT       1260                                                                          - ATAAAATAGA ATAAAATAAA GTGACTAAAA ATTAAACAAA TACCCTTTAA GA - #AATTAAAA       1320                                                                          - AAACTAAGGA AACATTTTTC TTGTTTCGAG TAGATAATGC CAGCCTGTTA AA - #CGCCGTCG       1380                                                                          - ACGAGTCTAA CGGACACCAA CCAGCGAACC AGCAGCGTCG CGTCGGGCCA AG - #CGAAGCAG       1440                                                                          - ACGGCACGGC ATCTCTGTCG CTGCCTCTGG ACCCCTCTCG AGAGTTCCGC TC - #CACCGTTG       1500                                                                          - GACTTGCTCC GCTGTCGGCA TCCAGAAATT GCGTGGCGGA GCGGCAGACG TG - #AGCCGGCA       1560                                                                          - CGGCAGGCGG CCTCCTCCTC CTCTCACGGC ACGGCAGCTA CGGGGGATTC CT - #TTCCCACC       1620                                                                          - GCTCCTACTA GAACTAGTGG ATCCTATGCG TATGGTATGA CGTGTGTTCA AG - #ATGATGAC       1680                                                                          - TTCAAACCTA CCTATGACGT ATGGTATGAC GTGTGTCGAC TGATGACTTA GA - #TCCACTCG       1740                                                                          - AGCGGCTATA AATACGTACC TACGCACCCT GCGCTACCAT CCCTAGAGCT GC - #ATGCTTAT       1800                                                                          #        1807                                                                 - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2620 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - TCTAGAGATA ATGAGCATTG CATGTCTAAG TTATAAAAAA TTACCACATA TT - #TTTTTTGT         60                                                                          - CACACTTGTT TGAAGTGCAG TTTATCTATC TTTATACATA TATTTAAACT TT - #ACTCTACG        120                                                                          - AATAATATAA TCTATAGTAC TACAATAATA TCAGTGTTTT AGAGAATCAT AT - #AAATGAAC        180                                                                          - AGTTAGACAT GGTCTAAAGG ACAATTGAGT ATTTTGACAA CAGGACTCTA CA - #GTTTTATC        240                                                                          - TTTTTAGTGT GCATGTGTTC TCCTTTTTTT TTGCAAATAG CTTCACCTAT AT - #AATACTTC        300                                                                          - ATCCATTTTA TTAGTACATC CATTTAGGGT TTAGGGTTAA TGGTTTTTAT AG - #ACTAATTT        360                                                                          - TTTTAGTACA TCTATTTTAT TCTATTTTAG CCTCTAAATT AAGAAAACTA AA - #ACTCTATT        420                                                                          - TTAGTTTTTT TATTTAATAA TTTAGATATA AAATAGAATA AAATAAAGTG AC - #TAAAAATT        480                                                                          - AAACAAATAC CCTTTAAGAA ATTAAAAAAA CTAAGGAAAC ATTTTTCTTG TT - #TCGAGTAG        540                                                                          - ATAATGCCAG CCTGTTAAAC GCCGTCGACG AGTCTAACGG ACACCAACCA GC - #GAACCAGC        600                                                                          - AGCGTCGCGT CGGGCCAAGC GAAGCAGACG GCACGGCATC TCTGTCGCTG CC - #TCTGGACC        660                                                                          - CCTCTCGAGA GTTCCGCTCC ACCGTTGGAC TTGCTCCGCT GTCGGCATCC AG - #AAATTGCG        720                                                                          - TGGCGGAGCG GCAGACGTGA GCCGGCACGG CAGGCGGCCT CCTCCTCCTC TC - #ACGGCACG        780                                                                          - GCAGCTACGG GGGATTCCTT TCCCACCGCT CCTACTAGAG ATAATGAGCA TT - #GCATGTCT        840                                                                          - AAGTTATAAA AAATTACCAC ATATTTTTTT TGTCACACTT GTTTGAAGTG CA - #GTTTATCT        900                                                                          - ATCTTTATAC ATATATTTAA ACTTTACTCT ACGAATAATA TAATCTATAG TA - #CTACAATA        960                                                                          - ATATCAGTGT TTTAGAGAAT CATATAAATG AACAGTTAGA CATGGTCTAA AG - #GACAATTG       1020                                                                          - AGTATTTTGA CAACAGGACT CTACAGTTTT ATCTTTTTAG TGTGCATGTG TT - #CTCCTTTT       1080                                                                          - TTTTTGCAAA TAGCTTCACC TATATAATAC TTCATCCATT TTATTAGTAC AT - #CCATTTAG       1140                                                                          - GGTTTAGGGT TAATGGTTTT TATAGACTAA TTTTTTTAGT ACATCTATTT TA - #TTCTATTT       1200                                                                          - TAGCCTCTAA ATTAAGAAAA CTAAAACTCT ATTTTAGTTT TTTTATTTAA TA - #ATTTAGAT       1260                                                                          - ATAAAATAGA ATAAAATAAA GTGACTAAAA ATTAAACAAA TACCCTTTAA GA - #AATTAAAA       1320                                                                          - AAACTAAGGA AACATTTTTC TTGTTTCGAG TAGATAATGC CAGCCTGTTA AA - #CGCCGTCG       1380                                                                          - ACGAGTCTAA CGGACACCAA CCAGCGAACC AGCAGCGTCG CGTCGGGCCA AG - #CGAAGCAG       1440                                                                          - ACGGCACGGC ATCTCTGTCG CTGCCTCTGG ACCCCTCTCG AGAGTTCCGC TC - #CACCGTTG       1500                                                                          - GACTTGCTCC GCTGTCGGCA TCCAGAAATT GCGTGGCGGA GCGGCAGACG TG - #AGCCGGCA       1560                                                                          - CGGCAGGCGG CCTCCTCCTC CTCTCACGGC ACGGCAGCTA CGGGGGATTC CT - #TTCCCACC       1620                                                                          - GCTCCTACTA GAGATAATGA GCATTGCATG TCTAAGTTAT AAAAAATTAC CA - #CATATTTT       1680                                                                          - TTTTGTCACA CTTGTTTGAA GTGCAGTTTA TCTATCTTTA TACATATATT TA - #AACTTTAC       1740                                                                          - TCTACGAATA ATATAATCTA TAGTACTACA ATAATATCAG TGTTTTAGAG AA - #TCATATAA       1800                                                                          - ATGAACAGTT AGACATGGTC TAAAGGACAA TTGAGTATTT TGACAACAGG AC - #TCTACAGT       1860                                                                          - TTTATCTTTT TAGTGTGCAT GTGTTCTCCT TTTTTTTTGC AAATAGCTTC AC - #CTATATAA       1920                                                                          - TACTTCATCC ATTTTATTAG TACATCCATT TAGGGTTTAG GGTTAATGGT TT - #TTATAGAC       1980                                                                          - TAATTTTTTT AGTACATCTA TTTTATTCTA TTTTAGCCTC TAAATTAAGA AA - #ACTAAAAC       2040                                                                          - TCTATTTTAG TTTTTTTATT TAATAATTTA GATATAAAAT AGAATAAAAT AA - #AGTGACTA       2100                                                                          - AAAATTAAAC AAATACCCTT TAAGAAATTA AAAAAACTAA GGAAACATTT TT - #CTTGTTTC       2160                                                                          - GAGTAGATAA TGCCAGCCTG TTAAACGCCG TCGACGAGTC TAACGGACAC CA - #ACCAGCGA       2220                                                                          - ACCAGCAGCG TCGCGTCGGG CCAAGCGAAG CAGACGGCAC GGCATCTCTG TC - #GCTGCCTC       2280                                                                          - TGGACCCCTC TCGAGAGTTC CGCTCCACCG TTGGACTTGC TCCGCTGTCG GC - #ATCCAGAA       2340                                                                          - ATTGCGTGGC GGAGCGGCAG ACGTGAGCCG GCACGGCAGG CGGCCTCCTC CT - #CCTCTCAC       2400                                                                          - GGCACGGCAG CTACGGGGGA TTCCTTTCCC ACCGCTCCTA CTAGAACTAG TG - #GATCCTAT       2460                                                                          - GCGTATGGTA TGACGTGTGT TCAAGATGAT GACTTCAAAC CTACCTATGA CG - #TATGGTAT       2520                                                                          - GACGTGTGTC GACTGATGAC TTAGATCCAC TCGAGCGGCT ATAAATACGT AC - #CTACGCAC       2580                                                                          #  2620            TAGA GCTGCATGCT TATTTTTACA                                 - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3433 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - TCTAGAGATA ATGAGCATTG CATGTCTAAG TTATAAAAAA TTACCACATA TT - #TTTTTTGT         60                                                                          - CACACTTGTT TGAAGTGCAG TTTATCTATC TTTATACATA TATTTAAACT TT - #ACTCTACG        120                                                                          - AATAATATAA TCTATAGTAC TACAATAATA TCAGTGTTTT AGAGAATCAT AT - #AAATGAAC        180                                                                          - AGTTAGACAT GGTCTAAAGG ACAATTGAGT ATTTTGACAA CAGGACTCTA CA - #GTTTTATC        240                                                                          - TTTTTAGTGT GCATGTGTTC TCCTTTTTTT TTGCAAATAG CTTCACCTAT AT - #AATACTTC        300                                                                          - ATCCATTTTA TTAGTACATC CATTTAGGGT TTAGGGTTAA TGGTTTTTAT AG - #ACTAATTT        360                                                                          - TTTTAGTACA TCTATTTTAT TCTATTTTAG CCTCTAAATT AAGAAAACTA AA - #ACTCTATT        420                                                                          - TTAGTTTTTT TATTTAATAA TTTAGATATA AAATAGAATA AAATAAAGTG AC - #TAAAAATT        480                                                                          - AAACAAATAC CCTTTAAGAA ATTAAAAAAA CTAAGGAAAC ATTTTTCTTG TT - #TCGAGTAG        540                                                                          - ATAATGCCAG CCTGTTAAAC GCCGTCGACG AGTCTAACGG ACACCAACCA GC - #GAACCAGC        600                                                                          - AGCGTCGCGT CGGGCCAAGC GAAGCAGACG GCACGGCATC TCTGTCGCTG CC - #TCTGGACC        660                                                                          - CCTCTCGAGA GTTCCGCTCC ACCGTTGGAC TTGCTCCGCT GTCGGCATCC AG - #AAATTGCG        720                                                                          - TGGCGGAGCG GCAGACGTGA GCCGGCACGG CAGGCGGCCT CCTCCTCCTC TC - #ACGGCACG        780                                                                          - GCAGCTACGG GGGATTCCTT TCCCACCGCT CCTACTAGAG ATAATGAGCA TT - #GCATGTCT        840                                                                          - AAGTTATAAA AAATTACCAC ATATTTTTTT TGTCACACTT GTTTGAAGTG CA - #GTTTATCT        900                                                                          - ATCTTTATAC ATATATTTAA ACTTTACTCT ACGAATAATA TAATCTATAG TA - #CTACAATA        960                                                                          - ATATCAGTGT TTTAGAGAAT CATATAAATG AACAGTTAGA CATGGTCTAA AG - #GACAATTG       1020                                                                          - AGTATTTTGA CAACAGGACT CTACAGTTTT ATCTTTTTAG TGTGCATGTG TT - #CTCCTTTT       1080                                                                          - TTTTTGCAAA TAGCTTCACC TATATAATAC TTCATCCATT TTATTAGTAC AT - #CCATTTAG       1140                                                                          - GGTTTAGGGT TAATGGTTTT TATAGACTAA TTTTTTTAGT ACATCTATTT TA - #TTCTATTT       1200                                                                          - TAGCCTCTAA ATTAAGAAAA CTAAAACTCT ATTTTAGTTT TTTTATTTAA TA - #ATTTAGAT       1260                                                                          - ATAAAATAGA ATAAAATAAA GTGACTAAAA ATTAAACAAA TACCCTTTAA GA - #AATTAAAA       1320                                                                          - AAACTAAGGA AACATTTTTC TTGTTTCGAG TAGATAATGC CAGCCTGTTA AA - #CGCCGTCG       1380                                                                          - ACGAGTCTAA CGGACACCAA CCAGCGAACC AGCAGCGTCG CGTCGGGCCA AG - #CGAAGCAG       1440                                                                          - ACGGCACGGC ATCTCTGTCG CTGCCTCTGG ACCCCTCTCG AGAGTTCCGC TC - #CACCGTTG       1500                                                                          - GACTTGCTCC GCTGTCGGCA TCCAGAAATT GCGTGGCGGA GCGGCAGACG TG - #AGCCGGCA       1560                                                                          - CGGCAGGCGG CCTCCTCCTC CTCTCACGGC ACGGCAGCTA CGGGGGATTC CT - #TTCCCACC       1620                                                                          - GCTCCTACTA GAGATAATGA GCATTGCATG TCTAAGTTAT AAAAAATTAC CA - #CATATTTT       1680                                                                          - TTTTGTCACA CTTGTTTGAA GTGCAGTTTA TCTATCTTTA TACATATATT TA - #AACTTTAC       1740                                                                          - TCTACGAATA ATATAATCTA TAGTACTACA ATAATATCAG TGTTTTAGAG AA - #TCATATAA       1800                                                                          - ATGAACAGTT AGACATGGTC TAAAGGACAA TTGAGTATTT TGACAACAGG AC - #TCTACAGT       1860                                                                          - TTTATCTTTT TAGTGTGCAT GTGTTCTCCT TTTTTTTTGC AAATAGCTTC AC - #CTATATAA       1920                                                                          - TACTTCATCC ATTTTATTAG TACATCCATT TAGGGTTTAG GGTTAATGGT TT - #TTATAGAC       1980                                                                          - TAATTTTTTT AGTACATCTA TTTTATTCTA TTTTAGCCTC TAAATTAAGA AA - #ACTAAAAC       2040                                                                          - TCTATTTTAG TTTTTTTATT TAATAATTTA GATATAAAAT AGAATAAAAT AA - #AGTGACTA       2100                                                                          - AAAATTAAAC AAATACCCTT TAAGAAATTA AAAAAACTAA GGAAACATTT TT - #CTTGTTTC       2160                                                                          - GAGTAGATAA TGCCAGCCTG TTAAACGCCG TCGACGAGTC TAACGGACAC CA - #ACCAGCGA       2220                                                                          - ACCAGCAGCG TCGCGTCGGG CCAAGCGAAG CAGACGGCAC GGCATCTCTG TC - #GCTGCCTC       2280                                                                          - TGGACCCCTC TCGAGAGTTC CGCTCCACCG TTGGACTTGC TCCGCTGTCG GC - #ATCCAGAA       2340                                                                          - ATTGCGTGGC GGAGCGGCAG ACGTGAGCCG GCACGGCAGG CGGCCTCCTC CT - #CCTCTCAC       2400                                                                          - GGCACGGCAG CTACGGGGGA TTCCTTTCCC ACCGCTCCTA CTAGAGATAA TG - #AGCATTGC       2460                                                                          - ATGTCTAAGT TATAAAAAAT TACCACATAT TTTTTTTGTC ACACTTGTTT GA - #AGTGCAGT       2520                                                                          - TTATCTATCT TTATACATAT ATTTAAACTT TACTCTACGA ATAATATAAT CT - #ATAGTACT       2580                                                                          - ACAATAATAT CAGTGTTTTA GAGAATCATA TAAATGAACA GTTAGACATG GT - #CTAAAGGA       2640                                                                          - CAATTGAGTA TTTTGACAAC AGGACTCTAC AGTTTTATCT TTTTAGTGTG CA - #TGTGTTCT       2700                                                                          - CCTTTTTTTT TGCAAATAGC TTCACCTATA TAATACTTCA TCCATTTTAT TA - #GTACATCC       2760                                                                          - ATTTAGGGTT TAGGGTTAAT GGTTTTTATA GACTAATTTT TTTAGTACAT CT - #ATTTTATT       2820                                                                          - CTATTTTAGC CTCTAAATTA AGAAAACTAA AACTCTATTT TAGTTTTTTT AT - #TTAATAAT       2880                                                                          - TTAGATATAA AATAGAATAA AATAAAGTGA CTAAAAATTA AACAAATACC CT - #TTAAGAAA       2940                                                                          - TTAAAAAAAC TAAGGAAACA TTTTTCTTGT TTCGAGTAGA TAATGCCAGC CT - #GTTAAACG       3000                                                                          - CCGTCGACGA GTCTAACGGA CACCAACCAG CGAACCAGCA GCGTCGCGTC GG - #GCCAAGCG       3060                                                                          - AAGCAGACGG CACGGCATCT CTGTCGCTGC CTCTGGACCC CTCTCGAGAG TT - #CCGCTCCA       3120                                                                          - CCGTTGGACT TGCTCCGCTG TCGGCATCCA GAAATTGCGT GGCGGAGCGG CA - #GACGTGAG       3180                                                                          - CCGGCACGGC AGGCGGCCTC CTCCTCCTCT CACGGCACGG CAGCTACGGG GG - #ATTCCTTT       3240                                                                          - CCCACCGCTC CTACTAGAAC TAGTGGATCC TATGCGTATG GTATGACGTG TG - #TTCAAGAT       3300                                                                          - GATGACTTCA AACCTACCTA TGACGTATGG TATGACGTGT GTCGACTGAT GA - #CTTAGATC       3360                                                                          - CACTCGAGCG GCTATAAATA CGTACCTACG CACCCTGCGC TACCATCCCT AG - #AGCTGCAT       3420                                                                          #    3433                                                                     __________________________________________________________________________

What is claimed is:
 1. A synthetic DNA promoter sequence functional in aplant cell, said promoter sequence comprising a TATA motif,atranscription start site, and a region between said TATA motif and saidstart site that is at least 64% GC-rich; wherein said region is not aregion between a TATA motif and a transcription start site of nativemaize ubiquitin promoter, and whereinsaid promoter sequence is set forthin SEQ ID NO:10.
 2. A synthetic DNA promoter sequence functional in aplant cell, said promoter sequence comprising a TATA motif,atranscription start site, and a region between said TATA motif and saidstart site that is at least 64% GC-rich; wherein said region is not aregion between a TATA motif and a transcription start site of nativemaize ubiquitin promoter, and whereinsaid promoter sequence is set forthin SEQ ID NO:1.
 3. An expression cassette comprising a syntheticpromoter comprising a TATA motif,a transcription start site and a regionbetween said TATA motif and said start site that is at least 64% GCrich, a structural gene operatively linked to said promoter, and atranscription end site polyadenylation signal; wherein said region isnot a region between a TATA motif and a transcription start site ofnative maize ubiquitin promoter, and whereinsequence of said promoter isset forth in SEQ ID NO:1.
 4. An expression cassette comprising asynthetic promoter comprising a TATA motif,a transcription start siteand a region between said TATA motif and said start site that is atleast 64% GC rich, a structural gene operatively linked to saidpromoter, and a transcription end site polyadenylation signal;whereinsaid region is not a region between a TATA motif and atranscription start site of native maize ubiquitin promoter, and whereinsequence of said promoter is set forth in SEQ ID NO:10.
 5. An expressioncassette comprising a synthetic promoter comprising a TATA motif,atranscription start site and a region between said TATA motif and saidstart site that is at least 64% GC rich, a structural gene operativelylinked to said promoter, a transcription end site polyadenylationsignal, and an upstream element operatively linked to said promoter sothat transcription is enhanced; whereinsaid region is not a regionbetween a TATA motif and a transcription start site of native maizeubiquitin promoter; and wherein sequence of said upstream element is setforth in SEQ ID NO:2.
 6. A nucleic acid vector comprising the promoterof any of claims 2 or 3 operatively linked to a structural gene and anupstream element operatively linked to said promoter, whereinsequence ofsaid upstream element is set forth in SEQ ID NO:2.
 7. A syntheticupstream element having a sequence set forth in SEQ ID NO:2.
 8. Anexpression cassette comprising:a promoter sequence; a structural geneoperatively linked to said promoter sequence; a polyadenylation signal;and a synthetic upstream element comprising SEQ ID NO:2 operativelylinked to said promoter so that expression is enhanced.
 9. Theexpression cassette of claim 8, wherein said synthetic promoter sequenceis SEQ ID NO:2.
 10. A nucleic acid vector comprising the expressioncassette of claim
 8. 11. The vector of claim 10, wherein said syntheticupstream element is SEQ ID NO:2.
 12. A prokaryotic or eukaryotic hostcell transformed with the vector of claim
 10. 13. An isolated nucleotidesequence comprising a DNA enhancer sequence comprising the nucleotidesequence set forth in SEQ ID No:
 5. 14. A nucleotide sequence comprisinga promoter construct, said construct comprising in operable linkage acore promoter sequence and a Ubi-1 UAR, wherein said Ubi-1 UAR is amaize Ubi UAR comprising the sequence set forth in SEQ ID No:
 13. 15. Anexpression cassette comprising in operable linkage a core promotersequence,a Ubi UAR operably linked upstream to said core promoter toform a synthetic promoter construct, a nucleotide sequence of interestoperably linked to said synthetic promoter, and a polyadenylationsignal; whereinsaid Ubi-1 UAR comprises the sequence set forth in SEQ IDNo:13.